Progress in studies of enzyme mechanisms has slowed in recent years, in part because investigators have failed to clearly define all of the important questions that must be addressed in order to move towards final conclusions about these reaction mechanisms. Many of the studies described in this grant are designed to leverage the potential for creative work directed towards answering the following question: How do enzymes achieve their specificity in transition state (TS) binding? This potential has been harnessed in studies in Buffalo on the mechanism of action of triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase (OMPDC) and glycerol 3-phosphate dehydrogenase (GPDH). These enzymes undergo dianion-driven conformational changes from loose, unliganded, open enzymes to stiff, structured, catalytically active closed forms, which act as ?switches? that turn on the expression of tight transition state binding interactions. Four key question are addressed in this grant, with the goals of generalizing earlier conclusions from TIM, OMPDC, and GPDH to other enzymes, and of initiating new studies to develop novel inhibitors of TIM and OMPDC from pathogenic organisms. Key Question 1: What other protein catalysts utilize binding interactions of their nonreacting substrate fragments to drive enzyme-activating conformational changes? These experiments will probe whether the binding energy from the adenosine ring of the substrate for adenylate kinase, or from the NAD cofactor of the substrate for alcohol dehydrogenase, which drive conformational changes during catalysis by these enzymes, act as a switch to turn on tight transition state binding interactions. Key Question 2: What interactions between the catalytic and activation sites of TIM, OMPDC and GPDH enable utilization of the intrinsic substrate binding energy for catalysis? Experiments are described to characterize a network of amino acid side chains involved in catalysis by GPDH, and to characterize the mechanism for activation of OMPDC by the utilization of binding interactions between the enzyme and the ribosyl hydroxyl groups of the substrates orotidine 5'-monophosphate (OMP) and 5-F-OMP. Key Question 3: Are computational methods sufficiently advanced to model the effect of site-directed mutations on the activation barrier for reactions catalyzed by TIM and GPDH? Calculations will be carried out in collaboration with Professor Lynn Kamerlin in Uppsala, Sweden, to determine whether existing EVB methods are able to model the results of extensive mutagenesis studies on these enzymes, with the goal of expanding the limits of these computational methods. Key Question 4: What is the potential for selection of peptides that show species specificity for inhibition of TIM and OMPDC from pathogenic organisms? Experiments are proposed, in collaboration with Professor Hiroaki Suga at the University of Tokyo, Japan, to identify species-specific inhibitors for TIM from Trypanosoma brucei and OMPDC from Plasmodium falciparum, and to characterize the important inhibitor-protein interactions by X-ray crystallography and computational docking studies.